CN101353644B - Vascular endothelial cells, and preparation and use thereof - Google Patents
Vascular endothelial cells, and preparation and use thereof Download PDFInfo
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- CN101353644B CN101353644B CN200810222274XA CN200810222274A CN101353644B CN 101353644 B CN101353644 B CN 101353644B CN 200810222274X A CN200810222274X A CN 200810222274XA CN 200810222274 A CN200810222274 A CN 200810222274A CN 101353644 B CN101353644 B CN 101353644B
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Abstract
The invention discloses a vascular endothelial cell and a preparation method and application thereof; the method for separating the vascular endothelial cell disclosed by the invention comprises the following steps: II type collagenase is used for digesting umbilical vein of umbilical cord in vitro, cells are collected, then culture is carried out and adherent cells are obtained, namely the vascular endothelial cell. The separation method of the invention has simple operation and convenience and practicability; the obtained vascular endothelial cell has high purity, large amount, strong exogenic propagation and a plurality of times of passage, and not only has the functions of phagotrophy and tube formation, but also has the function of inhibiting progenitor cells to develop into dendritic cells; therefore, the invention establishes a stable technical system for the separation and culture of the vascular endothelial cell and lays the foundation for the study and the application of theendothelial cell.
Description
Technical field
The present invention relates to a kind of vascular endothelial cell and preparation method thereof and application.
Background technology
Vascular endothelial cell not only participates in regulating vascular permeability and coagulation process, and all has vital role in many processes such as immunomodulatory, transplant rejection, metastases.In these researchs were used, the separation of endotheliocyte and vitro culture were crucial.
At present, how from the human umbilical vein, to separate endotheliocyte.But the Human umbilical vein endothelial cells growth in vitro ability that obtains is relatively poor, and propagation is slow, former being commissioned to train foster being difficult for successfully, and going down to posterity to cultivate also to pass 3-4 generation.
Obtaining of endotheliocyte adopted machineries to scrape to follow the example of and enzyme digestion more.Machinery is scraped and followed the example of is that umbilical vein is cut off, scrape with the operation knife back etc. and get the separator tube that endotheliocyte or use have nylon mesh, but this method is difficult to the grasp dynamics, and the easy damaged endotheliocyte, and be mixed with other cell such as inoblast etc. more.Now many employing enzyme digestions, and select rational digestive ferment for use and grasp digestion time, be that one of key of endotheliocyte is obtained in success.Trypsinase, collagenase all can be used as the enzyme of digestion vascular endothelial cell, and collagenase is gentle, and effect is better.The selection of cell nutrient solution serum also is the key factor whether the decision vascular endothelial cell survives.High-quality various serum could guarantee the normal growth of endotheliocyte.
Summary of the invention
An object of the present invention is to provide a kind of method of separating blood vessel endothelium cell.
The method of separating blood vessel endothelium cell provided by the present invention is that collecting cell is cultivated again with the umbilical vein of the stripped umbilical cord of II Collagen Type VI enzymic digestion, and the attached cell of acquisition is vascular endothelial cell.
Wherein, the proportioning of described II Collagen Type VI enzyme and described stripped umbilical cord can be the long umbilical cord of 85U-342U:1cm, is preferably the long umbilical cord of 171U:1cm; The time of described digestion can be 10-30min, is preferably 10min.
Solution with described II Collagen Type VI enzyme digests, and the starting point concentration of described II Collagen Type VI enzyme solution can be 171U/ml-684U/ml, is preferably 342U/ml.
The substratum that uses in the described cultivation is to add horse serum in the IMDM substratum, foetal calf serum, bovine serum albumin, heparin sodium, vascular endothelial growth factor and Basic Fibroblast Growth Factor, the final concentration that makes horse serum is 5-15% (volumn concentration), the final concentration of foetal calf serum is 5-15% (volumn concentration), the final concentration of bovine serum albumin is 0.5-2% (a quality percentage composition), the final concentration of heparin sodium is 10-20ug/ml, the final concentration of vascular endothelial growth factor is 5-20ng/ml, the final concentration of Basic Fibroblast Growth Factor is 1-5ng/ml, the substratum that obtains.
Described substratum is preferably and adds horse serum, foetal calf serum, bovine serum albumin, heparin sodium, vascular endothelial growth factor and Basic Fibroblast Growth Factor in the IMDM substratum, the final concentration that makes horse serum is that the final concentration of 10% (volumn concentration), foetal calf serum is that the final concentration of 10% (volumn concentration), bovine serum albumin is that the final concentration of 1% (quality percentage composition), heparin sodium is that the final concentration of 10ug/ml, vascular endothelial growth factor is that the final concentration of 10ng/ml, Basic Fibroblast Growth Factor is 1ng/ml, the substratum that obtains.
Wherein, the IMDM substratum both can directly be bought from company, also can oneself prepare.
The composition of IMDM substratum: 1L IMDM substratum contains 17.7g IMDM powder, final concentration is the L-glutaminate (L-glutamine) of 2mmol/L and HEPES buffer and the 3.024gNaHCO that final concentration is 25mmol/L
3
Described culture condition can be 35-38 ℃, CO for temperature
2Concentration is that the time of 5-8% (volumn concentration), saturated humidity, cultivation is 3-4 days; Described temperature is preferably 37 ℃, described CO
2Concentration is preferably 5%.
In the culturing process in per generation, meter when cultivating beginning behind the cultivation 24h, changes once fresh above-mentioned cell culture medium, in time removing non-adherent cell, and stays adherent endotheliocyte.
Described cultivation also can comprise succeeding transfer culture.
Umbilical vein is specifically as follows human umbilical vein described in the aforesaid method.
The vascular endothelial cell that obtains with above-mentioned arbitrary described method also belongs to protection scope of the present invention.
Another object of the present invention provides a kind of method of cultivating above-mentioned cell.
Cell culture processes provided by the present invention is to be 35-38 ℃, CO in temperature
2Concentration is 5-8% (volumn concentration), cultivate described cell under the condition of saturated humidity, cultivating used substratum can be to add horse serum in the IMDM substratum, foetal calf serum, bovine serum albumin, heparin sodium, vascular endothelial growth factor and Basic Fibroblast Growth Factor, the final concentration that makes horse serum is 5-15% (volumn concentration), the final concentration of foetal calf serum is 5-15% (volumn concentration), the final concentration of bovine serum albumin is 0.5-2% (a quality percentage composition), the final concentration of heparin sodium is 10-20ug/ml, the final concentration of vascular endothelial growth factor is 5-20ng/ml, the final concentration of Basic Fibroblast Growth Factor is 1-5ng/ml, the substratum that obtains.
In the culture condition, described temperature is preferably 37 ℃, described CO
2Concentration is preferably 5% (volumn concentration).
Described substratum is preferably and adds horse serum, foetal calf serum, bovine serum albumin, heparin sodium, vascular endothelial growth factor and Basic Fibroblast Growth Factor in the IMDM substratum, the final concentration that makes horse serum is that the final concentration of 10% (volumn concentration), foetal calf serum is that the final concentration of 10% (volumn concentration), bovine serum albumin is that the final concentration of 1% (quality percentage composition), heparin sodium is that the final concentration of 10ug/ml, vascular endothelial growth factor is that the final concentration of 10ng/ml, Basic Fibroblast Growth Factor is 1ng/ml, the substratum that obtains.
The application of above-mentioned cell in the precursor cell bud into dendritic cell that suppresses dendritic cell also belongs to protection scope of the present invention.
Separation method of the present invention is simple, convenient and practical, resulting endotheliocyte purity height (go down to posterity cultivated for 2 generations after, purity can reach 95%, 96% or 97%), many (umbilical cord acquisition 3-4 * 10 of 20 cm long of quantity
6Individual cell), in-vitro multiplication vigorous, can repeatedly go down to posterity (can pass 8-10 generation at most, still keep original activity), not only have and engulf and become the pipe ability, also have the function that suppresses precursor cell bud into dendritic cell.And dendritic cell is immunoreactive initiating person, can reduce immune response by the growth that suppresses dendritic cell, thereby helps the treatment that reduces graft-vs-host reaction and help some autoimmune diseases.
Therefore, the present invention has set up the separation and the culture technique system of stable vascular endothelial cell, uses for the research of endotheliocyte and lays a good foundation.
Description of drawings
Fig. 1 is morphologic observation result in the endotheliocyte culturing process.
Fig. 2 is the streaming detected result of endotheliocyte.
Fig. 3 is engulfing of endotheliocyte and becomes the pipe experimental result.
Fig. 4 is the result of the inhibition precursor cell bud into dendritic cell of endotheliocyte.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
The separation of embodiment 1, endotheliocyte and cultivation
From the fresh umbilical vein of healthy term birth of newborn, separate endotheliocyte in this experiment.Elder generation is with under the fresh umbilical cord scissors of newborn infant during operation.
The raw material of using in this experiment is as follows: II Collagen Type VI enzyme (Gibco), IMDM substratum (Gibco, Cat.No.12200-036)), foetal calf serum (Hyclone), horse serum (Gibco), bovine serum albumin BSA (Fluka), vascular endothelial growth factor VEGF (Prepro Tech), Basic Fibroblast Growth Factor bFGF (Prepro Tech), heparin sodium (Shanghai Chemical Reagent Co., Ltd., Sinopharm Group).
II Collagen Type VI enzyme solution is to prepare with IMDM substratum dissolving II Collagen Type VI enzyme dry powder.
One, separation and cultivation under condition I
Described condition I is as follows: the starting point concentration of used II Collagen Type VI enzyme solution is 342U/ml, digests 10 minutes; Cell culture condition is 37 ℃, CO
2Concentration 5%, saturated humidity; Used endotheliocyte substratum is to add horse serum, foetal calf serum, bovine serum albumin, heparin sodium, vascular endothelial growth factor and Basic Fibroblast Growth Factor in the IMDM substratum, the final concentration that makes horse serum is that the final concentration of 10% (volumn concentration), foetal calf serum is that the final concentration of 10% (volumn concentration), bovine serum albumin is that the final concentration of 1% (quality percentage composition), heparin sodium is that the final concentration of 10ug/ml, vascular endothelial growth factor is that the final concentration of 10ng/ml, Basic Fibroblast Growth Factor is 1ng/ml, the substratum that obtains.
Get the fresh umbilical cord 20cm of healthy full-term normal delivery newborn infant, clean 2 times with cold PBS damping fluid (containing the heparin sodium that final concentration is 200ug/ml); Clamp an end of umbilical cord with aseptic mosquito forceps, extract the II Collagen Type VI enzyme solution injection umbilical vein (proportioning of enzyme and umbilical cord is the long umbilical cord of 171U:1cm) that the 10ml starting point concentration is 342U/ml with asepsis injector, clamp the other end of umbilical cord with aseptic mosquito forceps, put into the sterilization plate, add PBS (containing the heparin sodium that final concentration is 200ug/ml) and preserve moisture, 37 ℃ of digestion 10min; Umbilical cord one end is put into the centrifuge tube that contains the 2ml foetal calf serum, draw 6ml IMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF) with aseptic straw, the flushing umbilical vein, with the cell solution that obtains with the centrifugal 10min of the speed of 1000 commentaries on classics/min, the collecting cell precipitation, with 1mlIMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF) re-suspended cell, carry out cell counting, adjusting cell concn with IMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF) is 1 * 10
6Individual/ml.Get 24 porocyte culture plates (costa), every hole adds 100 μ l IMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF), and 900 μ l endotheliocyte substratum are added in every hole again, at 37 ℃, 5%CO
2, cultivate 24h under the saturated humidity condition; Inhale then and remove liquid in 24 orifice plates, PBS washes 2 times, and every hole adds 1ml endotheliocyte substratum, at 37 ℃, 5%CO
2, continue under the saturated humidity condition to cultivate 3 days, the attached cell of acquisition is vascular endothelial cell (be former generation vascular endothelial cell); The number of the primary cell that statistics obtains shows, the long umbilical cord separation of 20cm obtains 4 * 10
6Individual vascular endothelial cell.
With the above-mentioned attached cell of 0.125% tryptic digestion, reach in 6 orifice plates, under identical condition, continue to cultivate (going down to posterity for the first time) with identical endotheliocyte substratum, grow to 80% when merging to cell, use 0.125% trypsin digestion and cell again, counting, with cultivations (second pass generation) that continue to go down to posterity of a part of cell, another part cell presses 1 * 10
6The concentration of individual/ml is put into cells frozen storing liquid (every milliliter of frozen storing liquid contains 0.4ml IMDM substratum, 0.5ml foetal calf serum, 0.1ml DMSO), and it is frozen to put into liquid nitrogen container again;
Second pass is commissioned to train supports the cell obtain and proceed again to go down to posterity, passed for 10 generations altogether.
Get the second pass cell that foster (3 generation cell) obtain of being commissioned to train, detect its purity with flow cytometer.Purity is 97% as a result.
Two, separation and cultivation under condition II
Described condition II is as follows: the concentration of used II Collagen Type VI enzyme is 171U/ml, digests 20 minutes; Cell culture condition is 35 ℃, CO
2Concentration 6%, saturated humidity; Used endotheliocyte substratum is to add horse serum, foetal calf serum, bovine serum albumin, heparin sodium, vascular endothelial growth factor and Basic Fibroblast Growth Factor in the IMDM substratum, the final concentration that makes horse serum is that the final concentration of 5% (volumn concentration), foetal calf serum is that the final concentration of 5% (volumn concentration), bovine serum albumin is that the final concentration of 0.5% (quality percentage composition), heparin sodium is that the final concentration of 15ug/ml, vascular endothelial growth factor is that the final concentration of 5ng/ml, Basic Fibroblast Growth Factor is 3ng/ml, the substratum that obtains.
Get the fresh umbilical cord 20cm of healthy full-term normal delivery newborn infant, clean 2 times with cold PBS damping fluid (containing the heparin sodium that final concentration is 200ug/ml); Clamp an end of umbilical cord with aseptic mosquito forceps, the II Collagen Type VI enzyme solution that extracts 10ml171U/ml with asepsis injector injects umbilical vein (proportioning of enzyme and umbilical cord is the long umbilical cord of 85U:1cm), clamp the other end of umbilical cord with aseptic mosquito forceps, put into the sterilization plate, add PBS (containing the heparin sodium that final concentration is 200ug/ml) and preserve moisture, 37 ℃ of digestion 20min; Umbilical cord one end is put into the centrifuge tube that contains the 2ml foetal calf serum, draw 6ml IMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF) with aseptic straw, the flushing umbilical vein, with the cell solution that obtains with the centrifugal 10min of the speed of 1000 commentaries on classics/min, the collecting cell precipitation, with 1mlIMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF) re-suspended cell, carry out cell counting, adjusting cell concn with IMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF) is 1 * 10
6Individual/ml.Get 24 porocyte culture plates (costa), every hole adds 100 μ l IMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF), and 900 μ l endotheliocyte substratum are added in every hole again, at 35 ℃, 6%CO
2, cultivate 24h under the saturated humidity condition; Inhale then and remove liquid in 24 orifice plates, PBS washes 2 times, and every hole adds 1ml endotheliocyte substratum, at 37 ℃, 5%CO
2, continue under the saturated humidity condition to cultivate 2 days, the attached cell of acquisition is vascular endothelial cell (be former generation vascular endothelial cell); The number of the primary cell that statistics obtains shows, the long umbilical cord separation of 20cm obtains 3 * 10
6Each and every one vascular endothelial cell.
With the above-mentioned attached cell of 0.125% tryptic digestion, reach in 6 orifice plates, under identical condition, continue to cultivate (going down to posterity for the first time) with identical endotheliocyte substratum, grow to 80% when merging to cell, use 0.125% trypsin digestion and cell again, counting is with cultivations ((second pass generation) that continue to go down to posterity of a part of cell), another part cell presses 1 * 10
6The concentration of individual/ml is put into cells frozen storing liquid (every milliliter of frozen storing liquid contains 0.4ml IMDM substratum, 0.5ml foetal calf serum, 0.1ml DMSO), and it is frozen to put into liquid nitrogen container again;
Second pass is commissioned to train supports the cell obtain and proceed again to go down to posterity, passed for 8 generations altogether.
Get the second pass cell that foster (3 generation cell) obtain of being commissioned to train, detect its purity with flow cytometer.Purity is 95% as a result.
Three, separation and cultivation under condition III
Described condition III is as follows: the concentration of used II Collagen Type VI enzyme is 684U/ml, digests 30 minutes; Cell culture condition is 38 ℃, CO
2Concentration 8%, saturated humidity; Used endotheliocyte substratum is to add horse serum, foetal calf serum, bovine serum albumin, heparin sodium, vascular endothelial growth factor and Basic Fibroblast Growth Factor in the IMDM substratum, the final concentration that makes horse serum is that the final concentration of 15% (volumn concentration), foetal calf serum is that the final concentration of 15% (volumn concentration), bovine serum albumin is that the final concentration of 2% (quality percentage composition), heparin sodium is that the final concentration of 20ug/ml, vascular endothelial growth factor is that the final concentration of 20ng/ml, Basic Fibroblast Growth Factor is 5ng/ml, the substratum that obtains.
Get the fresh umbilical cord 20cm of healthy full-term normal delivery newborn infant, clean 2 times with cold PBS damping fluid (containing the heparin sodium that final concentration is 200ug/ml); Clamp an end of umbilical cord with aseptic mosquito forceps, the II Collagen Type VI enzyme solution that extracts 10ml684U/ml with asepsis injector injects umbilical vein (proportioning of enzyme and umbilical cord is the long umbilical cord of 342U:1cm), clamp the other end of umbilical cord with aseptic mosquito forceps, put into the sterilization plate, add PBS (containing the heparin sodium that final concentration is 200ug/ml) and preserve moisture, 37 ℃ of digestion 30min; Umbilical cord one end is put into the centrifuge tube that contains the 2ml foetal calf serum, draw 6ml IMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF) with aseptic straw, the flushing umbilical vein, with the cell solution that obtains with the centrifugal 10min of the speed of 1000 commentaries on classics/min, the collecting cell precipitation, with 1mlIMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF) re-suspended cell, carry out cell counting, adjusting cell concn with IMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF) is 1 * 10
6Individual/ml.Get 24 porocyte culture plates (costa), every hole adds 100 μ l IMDM substratum (not containing horse serum, foetal calf serum, BSA, heparin, VEGF and bFGF), and 900 μ l endotheliocyte substratum are added in every hole again, at 38 ℃, 8%CO
2, cultivate 24h under the saturated humidity condition; Inhale then and remove liquid in 24 orifice plates, PBS washes 2 times, and every hole adds 1ml endotheliocyte substratum, at 37 ℃, 5%CO
2, continue under the saturated humidity condition to cultivate 3 days, the attached cell of acquisition is vascular endothelial cell (be former generation vascular endothelial cell); The number of the primary cell that statistics obtains shows, the long umbilical cord separation of 20cm obtains 3.5 * 10
6Each and every one vascular endothelial cell.
With the above-mentioned attached cell of 0.125% tryptic digestion, reach in 6 orifice plates, under identical condition, continue to cultivate (going down to posterity for the first time) with identical endotheliocyte substratum, grow to 80% when merging to cell, use 0.125% trypsin digestion and cell again, counting is with cultivations ((second pass generation) that continue to go down to posterity of a part of cell), another part cell presses 1 * 10
6The concentration of individual/ml is put into cells frozen storing liquid (every milliliter of frozen storing liquid contains 0.4ml IMDM substratum, 0.5ml foetal calf serum, 0.1ml DMSO), and it is frozen to put into liquid nitrogen container again;
Second pass is commissioned to train supports the cell obtain and proceed again to go down to posterity, passed for 9 generations altogether.
Get the second pass cell that foster (3 generation cell) obtain of being commissioned to train, detect its purity with flow cytometer.Purity is 96% as a result.
The evaluation of embodiment 2, endotheliocyte
Each of getting that cell that cell that embodiment 1 one Central Plains generations obtains to going down to posterity for the 10th time, embodiment 1 two Central Plains generations obtains to going down to posterity for the 8th time, embodiment 1 three Central Plains generations obtains to going down to posterity for the 9th time carried out following identification experiment respectively for cell.
1, cellular form is observed
Each endotheliocyte all forms several clones in 24 orifice plates in process of growth, cell is the shuttle shape, when treating that cell is paved with 24 orifice plates, the cell chap shortens, and is cobblestone-appearance, the form of the primary cell that obtains among the embodiment 1 one (Figure 1A is the shuttle shape, and Figure 1B is a cobblestone-appearance) as shown in Figure 1 for example.Show that on form, the cell that the present invention obtains meets the morphological specificity of vascular endothelial cell.
2, flow cytometer detects its surface marker
Use following mouse-anti human monoclonal antibodies in this experiment: CD31-PE, CD73-PE, CD166-PE, CD34-FITC, HLA-DR-FITC, HLA-ABC-FITC, CD45-FITC, CD29-PE all are that BD company produces.
Cell counting respectively is divided into 5 pipes, every pipe 5 * 10
5Individual cell, centrifugal, collecting cell precipitation, every Guan Zaiyong 200 μ l PBS damping fluids are resuspended, wherein a tube cell in contrast, all the other four pipes are as experimental group, every kind of antibody of 10 μ l respectively in 4 experiment tubes is specific as follows:
First pipe: CD31-PE and CD34-FITC;
Second pipe: CD73-PE and HLA-DR-FITC;
The 3rd pipe: CD166-PE and HLA-ABC-FITC;
The 4th pipe: CD29-PE and CD45-FITC;
Each tube cell is all placed the lucifuge place, and incubated at room 20min adds that the PBS damping fluid is centrifugal to be washed one time, and every pipe adds 300 μ l0.5% Paraformaldehyde 96s again to be fixed, and 4 ℃ of preservations were carried out fluidic cell and detected in second day.
3 repetitions are established in experiment.The result shows, in all detected cells, have the above cell of 90-95% to show as CD31-PE, CD73-PE, HLA-ABC-FITC and the CD29-PE positive, CD34-FITC, HLA-DR-FITC, CD166-PE and CD45-FITC feminine gender meet the feature of vascular endothelial cell.Second pass for the detected result of the cell that obtains as shown in Figure 2 among the embodiment 1 one.
3, endotheliocyte engulfs and becomes pipe
(1) the Tip head of preparing one 48 porocyte culture plate (costa) and 20-200 μ l place-20 ℃ freezing; Matrigel (Sigma) places 4 ℃ to thaw.
(2) 48 orifice plates are got a hole and are spread glue with 30 μ l Matrigel, and the Tip head with precooling is swift in motion, in case Matrigel at room temperature solidifies the too fast glue face injustice that causes.Place 30min for 37 ℃, make gelling solid.
(3) cell counting gets 2 * 10
4Cell adds have been spread in the hole of Matrigel glue, adds endotheliocyte substratum to 200 μ l, and 37 ℃, 5%CO
2Saturated humidity is cultivated, and behind the 4h, adds 5 μ l Dil-Ac-LDL (Sigma), and 37 ℃, 5%CO
2Saturated humidity is cultivated 8h, and fluorescence microscope is engulfed and become the duct ligation fruit.
Experimental result shows that vascular endothelial cell of the present invention all can be engulfed Dil-Ac-LDL, all has into the pipe ability on Matrigel glue.The detected result (A is for engulfing the result, and B is for becoming duct ligation really) as shown in Figure 3 of the cell that obtains for the third time goes down to posterity among the embodiment 1 one.
4, endotheliocyte can suppress precursor cell (CD14
+Cell) grows to dendritic cell
(1) get stripped human peripheral leucocytes 10ml, the Fillco density gradient centrifugation separates mononuclearcell, and by the operation of person monocytic cell's separating kit (MACS) specification sheets, separating monocytic cell is CD14
+Cell (being the precursor cell of dendritic cell).
(2) endotheliocyte is inoculated in the hole in 6 orifice plates (Costa), and cell count is 4 * 10
5, 37 ℃, 5%CO
2Saturated humidity is cultivated 12h, adds 4 * 10 in this hole
6Monocyte, other gets a hole and adds 4 * 10
6Monocyte adds GM-CSF (Prepro Tech) respectively (10ng/ml) and (10ng/ml) 37 ℃ of IL-4 (Prepro Tech), 5%CO in every hole
2Saturated humidity is cultivated, and every other day partly changes once fresh endotheliocyte substratum, adds the GM-CSF and the IL-4 of same concentrations simultaneously.
(3) behind the cell cultures 7d, collecting cell, the expression of Flow cytometry CD1a (BD) and CD14 (BD).
Normal dendritic cell energy high expression level CD1a (Fig. 4 A) and CD83 (Fig. 4 B), but among the present invention, with CD14
+After cell (being the precursor cell of dendritic cell) is cultivated altogether with vascular endothelial cell of the present invention, CD1a of the cell expressing that obtains (Fig. 4 C) and CD83 (Fig. 4 D) obviously reduce, show that vascular endothelial cell of the present invention can suppress precursor cell bud into dendritic cell.The experimental result of the cell that obtains go down to posterity among the embodiment 1 one for the third time shown in Fig. 4 C and Fig. 4 D.
To sum up experiment shows, the cell that the present invention obtains is exactly vascular endothelial cell and the function that suppresses precursor cell bud into dendritic cell is arranged.
Claims (9)
1. vascular endothelial cell is divided into application in the dendritic cell in the vitro inhibition peripheral blood lymphocytes;
Described vascular endothelial cell separates as follows: with the umbilical vein of the stripped umbilical cord of II Collagen Type VI enzymic digestion, collecting cell is cultivated again, and the attached cell of acquisition is vascular endothelial cell;
The proportioning of described II Collagen Type VI enzyme and described stripped umbilical cord is the long umbilical cord of 85U-342U:1cm; The time of described digestion is 10-30min;
Solution with described II Collagen Type VI enzyme digests, and the starting point concentration of described II Collagen Type VI enzyme solution is 171U/ml-684U/ml.
2. application according to claim 1 is characterized in that: the proportioning of described II Collagen Type VI enzyme and described stripped umbilical cord is the long umbilical cord of 171U:1cm; The time of described digestion is 10min.
3. application according to claim 2 is characterized in that: the solution with described II Collagen Type VI enzyme digests, and the starting point concentration of described II Collagen Type VI enzyme solution is 342U/ml.
4. according to arbitrary described application among the claim 1-3, it is characterized in that: the substratum that uses in the described cultivation is to add horse serum in the IMDM substratum, foetal calf serum, bovine serum albumin, heparin sodium, vascular endothelial growth factor and Basic Fibroblast Growth Factor, the final concentration that makes horse serum is 5-15% (volumn concentration), the final concentration of foetal calf serum is 5-15% (volumn concentration), the final concentration of bovine serum albumin is 0.5-2% (a quality percentage composition), the final concentration of heparin sodium is 10-20ug/ml, the final concentration of vascular endothelial growth factor is 5-20ng/ml, the final concentration of Basic Fibroblast Growth Factor is 1-5ng/ml, the substratum that obtains.
5. application according to claim 4 is characterized in that: the final concentration that the final concentration of described horse serum is 10%, the final concentration of described foetal calf serum is 10%, the final concentration of described bovine serum albumin is 1%, the final concentration of described heparin sodium is 10ug/ml, described vascular endothelial growth factor is that the final concentration of 10ng/ml, described Basic Fibroblast Growth Factor is 1ng/ml.
6. according to arbitrary described application among the claim 1-3, it is characterized in that: the temperature of described cultivation is 35-38 ℃, CO
2Concentration is that 5-8% (volumn concentration), humidity are that saturated humidity, incubation time are 3-4 days.
7. application according to claim 6 is characterized in that: the temperature of described cultivation is 37 ℃, described CO
2Concentration is 5%.
8. application according to claim 7 is characterized in that: after described cultivation is carried out 24h from the outset, change once fresh described substratum.
9. application according to claim 8 is characterized in that: described cultivation also comprises succeeding transfer culture.
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| CN104974978B (en) * | 2015-08-05 | 2018-11-27 | 广州赛莱拉干细胞科技股份有限公司 | A kind of cultural method of Endothelial cell culture base and endothelial cell |
| CN105907704A (en) * | 2016-05-20 | 2016-08-31 | 山东省齐鲁干细胞工程有限公司 | Method for isolated culture of umbilical vein endothelial cell |
| CN106591221B (en) * | 2017-01-16 | 2020-10-02 | 北京京蒙高科干细胞技术有限公司 | Method for separating and amplifying human vein endothelial cells in vitro |
| CN110484491B (en) * | 2019-08-20 | 2022-03-18 | 北京京蒙高科干细胞技术有限公司 | Method for obtaining amniotic membrane and amniotic fluid derived endothelial progenitor cells and purification culture method thereof |
| CN111109248A (en) * | 2019-12-24 | 2020-05-08 | 武汉博士德生物工程有限公司 | Hybridoma cell cryopreservation liquid and 96-well plate cryopreservation recovery method |
| CN117990923B (en) * | 2023-11-20 | 2024-10-01 | 昆明市公安局 | Hemoglobin treatment fluid, blood trace species identification method and application |
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